The integrity of the lining of the airways relies on multiple functions of its epithelial stem cells. We hypothesize that the airway stem cell behavior is regulated by the cells and the forces that surrounds them, including other epithelial cells, subepithelial mesenchymal cells, and external mechanical forces exerted on the epithelium. How these varied components regulate the behavior of airway progenitor cells is largely unknown. Furthermore, basal stem cell ? niche interactions may explain the increasingly recognized heterogeneity within this progenitor cell population, including regional and tissue-level heterogeneity in basal stem cell behavior. Although progenitor cell ? niche interactions have been described with live imaging in other systems, the constant motion and challenging access of the respiratory system has hampered the development of platforms for live imaging of the airways at high resolution. We combined emerging stem cell biology and live imaging technologies to develop a novel airway explant live imaging platform. We imaged the maintenance and regeneration of the airway epithelium in a mouse airway explant, surrounded by all of the major components of its 3D multicomponent microenvironment, including the ECM and the mesenchyme. We present preliminary data on tracking individual stem cells during regeneration with two photon laser scanning microscopy, including visualization of stem cell differentiation with fluorescent reporters and analysis of ciliated cell function with novel video-rate imaging modalities. To define the airway stem cell microenvironment and to test its role in progenitor cells function, we visualize and putative cellular niche components and test their role by laser and toxin-mediated ablation. We propose to use cellular engraftment followed by application of mechanical forces to check the role of the microenvironment in establishing planar polarity, and to study the relationship of proliferation, migration and differentiation in collective cell migration in the airway epithelium.